RESUMO
SUMMARY: Comparisons of protein structures are critical for developing novel protein designs, annotating protein functions and predicting protein structure. The template modeling score (TM-score) is a widely used but computationally expensive measure of protein similarity that is applicable to a wide variety of structural biology problems. We introduce TMQuery-a continuously updated database containing over eight billion pre-computed TM-score values for every pair of proteins in the Protein Data Bank, allowing researchers to quickly query and download TM-scores via a web interface. AVAILABILITY AND IMPLEMENTATION: Publicly available at https://tmquery.gsk.com/.
Assuntos
Proteínas , Software , Conformação Proteica , Proteínas/química , Bases de Dados de ProteínasRESUMO
The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli. Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.
Assuntos
Nanopartículas , Neisseria meningitidis , Camundongos , Animais , Ferritinas , Antígenos de Bactérias , Antígenos Virais , Anticorpos Bloqueadores , Vacinas Combinadas , Nanopartículas/químicaRESUMO
The classical complement pathway is triggered when antigen-bound immunoglobulins bind to C1q through their Fc region. While C1q binds to a single Fc with low affinity, a higher avidity stable binding of two or more of C1q globular heads initiates the downstream reactions of the complement cascade ultimately resulting in bacteriolysis. Synergistic bactericidal activity has been demonstrated when monoclonal antibodies recognize nonoverlapping epitopes of the same antigen. The aim of the present work was to investigate the synergistic effect between antibodies directed toward different antigens. To this purpose, we investigated the bactericidal activity induced by combinations of monoclonal antibodies (mAbs) raised against factor H-binding protein (fHbp) and Neisserial Heparin-Binding Antigen (NHBA), two major antigens included in Bexsero, the vaccine against Meningococcus B, for prevention from this devastating disease in infants and adolescents. Collectively, our results show that mAbs recognizing different antigens can synergistically activate complement even when each single Mab is not bactericidal, reinforcing the evidence that cooperative immunity induced by antigen combinations can represent a remarkable added value of multicomponent vaccines. Our study also shows that the synergistic effect of antibodies is modulated by the nature of the respective epitopes, as well as by the antigen density on the bacterial cell surface.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Proteínas do Sistema Complemento/imunologia , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Transporte/imunologia , Fator H do Complemento/imunologia , Epitopos/imunologia , Neisseria meningitidis/imunologia , Ensaios de Anticorpos Bactericidas Séricos/métodosRESUMO
Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Neisseria meningitidis Sorogrupo B/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Variação Genética , Humanos , Espectrometria de Massas/métodos , Meningite Meningocócica/imunologia , Meningite Meningocócica/microbiologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/genética , Filogenia , Especificidade da EspécieRESUMO
Serogroup B Neisseria meningitidis (MenB) is a major cause of severe sepsis and invasive meningococcal disease, which is associated with 5-15% mortality and devastating long-term sequelae. Neisserial adhesin A (NadA), a trimeric autotransporter adhesin (TAA) that acts in adhesion to and invasion of host epithelial cells, is one of the three antigens discovered by genome mining that are part of the MenB vaccine that recently was approved by the European Medicines Agency. Here we present the crystal structure of NadA variant 5 at 2 Å resolution and transmission electron microscopy data for NadA variant 3 that is present in the vaccine. The two variants show similar overall topology with a novel TAA fold predominantly composed of trimeric coiled-coils with three protruding wing-like structures that create an unusual N-terminal head domain. Detailed mapping of the binding site of a bactericidal antibody by hydrogen/deuterium exchange MS shows that a protective conformational epitope is located in the head of NadA. These results provide information that is important for elucidating the biological function and vaccine efficacy of NadA.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Mapeamento de Epitopos/métodos , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Sítios de Ligação de Anticorpos/genética , Sítios de Ligação de Anticorpos/imunologia , Cristalografia por Raios X , Medição da Troca de Deutério , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Neisseria meningitidis Sorogrupo B/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Multimerização Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray , TemperaturaRESUMO
The production of capsular polysaccharides (CPS) or secreted exopolysaccharides is ubiquitous in bacteria, and the Wzy pathway constitutes a prototypical mechanism to produce these structures. Despite the differences in polysaccharide composition among species, a group of proteins involved in this pathway is well conserved. Streptococcus agalactiae (group B Streptococcus; GBS) produces a CPS that represents the main virulence factor of the bacterium and is a prime target in current vaccine development. We used this human pathogen to investigate the roles and potential interdependencies of the conserved proteins CpsABCD encoded in the cps operon, by developing knock-out and functional mutant strains. The mutant strains were examined for CPS quantity, size, and attachment to the cell surface as well as CpsD phosphorylation. We observed that CpsB, -C, and -D compose a phosphoregulatory system where the CpsD autokinase phosphorylates its C-terminal tyrosines in a CpsC-dependent manner. These Tyr residues are also the target of the cognate CpsB phosphatase. An interaction between CpsD and CpsC was observed, and the phosphorylation state of CpsD influenced the subsequent action of CpsC. The CpsC extracellular domain appeared necessary for the production of high molecular weight polysaccharides by influencing CpsA-mediated attachment of the CPS to the bacterial cell surface. In conclusion, although having no impact on cps transcription or the synthesis of the basal repeating unit, we suggest that these proteins are fine-tuning the last steps of CPS biosynthesis (i.e. the balance between polymerization and attachment to the cell wall).
Assuntos
Cápsulas Bacterianas/metabolismo , Proteínas de Bactérias/metabolismo , Óperon , Polímeros/metabolismo , Streptococcus agalactiae/metabolismo , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/ultraestrutura , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Immunoblotting , Camundongos , Microscopia Imunoeletrônica , Mutação , Fosforilação , Polissacarídeos Bacterianos/metabolismo , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Estreptocócicas/microbiologia , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidade , Tirosina/genética , Tirosina/metabolismo , Fatores de Virulência/genéticaRESUMO
Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with â¼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Lipídeo A/imunologia , Shigella/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Acilação/imunologia , Aciltransferases/genética , Aciltransferases/metabolismo , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Humanos , Lipídeo A/análise , Lipídeo A/metabolismo , Microscopia Eletrônica de Transmissão , Monócitos/imunologia , Monócitos/metabolismo , Mutação , Shigella/genética , Shigella/metabolismo , Shigella flexneri/genética , Shigella flexneri/imunologia , Shigella flexneri/metabolismo , Shigella sonnei/genética , Shigella sonnei/imunologia , Shigella sonnei/metabolismo , Transdução de Sinais/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Adjuvants are substances that enhance adaptive immune responses when formulated in a vaccine. Alum and MF59 are two vaccine adjuvants licensed for human vaccination. Their mode of action has not been completely elucidated. Here we show the first ultrastructural visualization of Alum and MF59 interaction with immune cells in vitro and in vivo. We observed that Alum is engulfed by cells as inclusions of laminae that are detectable within draining lymph nodes. MF59 is instead engulfed by cells in vitro as low-electron-dense lipid-like inclusions that display a vesicle pattern, as confirmed by confocal microscopy using fluorescently labeled MF59. However, lipid-like inclusions with different high- and low-electron-dense content are detected within cells of draining lymph nodes when injecting MF59. As high-electron-dense lipid-like inclusions are also detected upon injection of Alum, our results suggest that the low-electron-dense inclusions are formed by engulfed MF59, whereas the high-electron-dense inclusions are proper lipid inclusions. Thus, we demonstrated that vaccine adjuvants are engulfed as inclusions by lymph node cells and hypothesize that adjuvant treatment may modify lipid metabolism.
Assuntos
Adjuvantes Imunológicos/farmacocinética , Compostos de Alúmen/farmacocinética , Polissorbatos/farmacocinética , Esqualeno/farmacocinética , Adjuvantes Imunológicos/administração & dosagem , Compostos de Alúmen/administração & dosagem , Animais , Endocitose , Corpos de Inclusão/ultraestrutura , Camundongos Endogâmicos C57BL , Microscopia , Polissorbatos/administração & dosagem , Esqualeno/administração & dosagemRESUMO
Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 microm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.
Assuntos
Fímbrias Bacterianas/metabolismo , Proteínas Recombinantes/química , Streptococcus pneumoniae/metabolismo , Animais , Aderência Bacteriana , Proteínas de Bactérias/química , Western Blotting , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Transmissão/métodos , Mutação , Conformação ProteicaRESUMO
We previously reported finding the RNA of a type K human endogenous retrovirus, HERV-K (HML-2), at high titers in the plasma of HIV-1-infected and cancer patients (R. Contreras-Galindo et al., J. Virol. 82:9329-9236, 2008.). The extent to which the HERV-K (HML-2) proviruses become activated and the nature of their activated viral RNAs remain important questions. Therefore, we amplified and sequenced the full-length RNA of the env gene of the type 1 and 2 HERV-K (HML-2) viruses collected from the plasma of seven HIV-1-infected patients over a period of 1 to 3 years and from five breast cancer patients in order to reconstruct the genetic evolution of these viruses. HERV-K (HML-2) RNA was found in plasma fractions of HIV-1 patients at a density of â¼1.16 g/ml that contained both immature and correctly processed HERV-K (HML-2) proteins and virus-like particles that were recognized by anti-HERV-K (HML-2) antibodies. RNA sequences from novel HERV-K (HML-2) proviruses were discovered, including K111, which is specifically active during HIV-1 infection. Viral RNA arose from complete proviruses and proviruses devoid of a 5' long terminal repeat, suggesting that the expression of HERV-K (HML-2) RNA in these patients may involve sense and antisense transcription. In HIV-1-infected individuals, the HERV-K (HML-2) viral RNA showed evidence of frequent recombination, accumulation of synonymous rather than nonsynonymous mutations, and conserved N-glycosylation sites, suggesting that some of the HERV-K (HML-2) viral RNAs have undergone reverse transcription and are under purifying selection. In contrast, HERV-K (HML-2) RNA sequences found in the blood of breast cancer patients showed no evidence of recombination and exhibited only sporadic viral mutations. This study suggests that HERV-K (HML-2) is active in HIV-1-infected patients, and the resulting RNA message reveals previously undiscovered HERV-K (HML-2) genomic sequences.
Assuntos
Retrovirus Endógenos/genética , Infecções por HIV/virologia , HIV-1/genética , RNA Viral/genética , Retrovirus Endógenos/classificação , Retrovirus Endógenos/metabolismo , Genoma Viral , Infecções por HIV/sangue , HIV-1/classificação , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Filogenia , RNA Viral/sangue , RNA Viral/metabolismo , Recombinação Genética , Transcrição Reversa , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.
Assuntos
Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Multimerização Proteica , Streptococcus pneumoniae , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Cristalização , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Ligação de Hidrogênio , Fibras Minerais , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Multimerização Proteica/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.
Assuntos
Proteínas de Fímbrias/química , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/química , Adesão Celular , Modelos Animais de Doenças , Epitopos/química , Proteínas de Fímbrias/genética , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Sepse/metabolismoRESUMO
In group B Streptococcus (GBS), 3 structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular-weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies, and in vivo mutagenesis, we performed a broad characterization of GBS sortase C1 of pilus island 2a. The high-resolution X-ray structure of the enzyme revealed that the active site, into the ß-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the "lid." We show that the catalytic triad and the predicted N- and C-terminal transmembrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies, we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild-type protein.
Assuntos
Aminoaciltransferases/química , Aminoaciltransferases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Fímbrias Bacterianas/enzimologia , Regulação Bacteriana da Expressão Gênica/fisiologia , Streptococcus agalactiae/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Cristalografia por Raios X , Cisteína Endopeptidases/metabolismo , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Plasmídeos , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Alinhamento de Sequência , Streptococcus agalactiae/genéticaRESUMO
Moraxella catarrhalis is an important and common respiratory pathogen that can cause Otitis Media, Community Acquired Pneumonia, and has been associated with an increased risk of exacerbations in chronic obstructive pulmonary disease in adults, leading to morbidity and mortality. Its ubiquitous surface protein A2 (UspA2) has been shown to interact with host structures and extracellular matrix proteins, suggesting a role at an early stage of infection and a contribution to bacterial serum resistance. The UspA proteins are homo-trimeric autotransporters that appear as a lollipop-shaped structure in electron micrographs. They are composed of an N-terminal head with adhesive properties, followed by a stalk, which ends by an amphipathic helix and a C-terminal membrane domain. The three family members UspA1, UspA2 and UspA2H, present different amino acid signatures both at the head and membrane-spanning regions. By combining electron microscopy, hydrogen deuterium exchange mass spectrometry and protein modeling, we identified a shared and repeated epitope recognized by FHUSPA2/10, a potent cross-bactericidal monoclonal antibody raised by UspA2 and deduced key amino acids involved in the binding. The finding strengthens the potential of UspA2 to be incorporated in a vaccine formulation against M. catarrhalis.
Assuntos
Antibacterianos , Anticorpos Monoclonais , Moraxella catarrhalis , Adulto , Humanos , Aminoácidos/metabolismo , Anticorpos Monoclonais/farmacologia , Proteínas da Membrana Bacteriana Externa/imunologia , Epitopos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Sistemas de Secreção Tipo V/metabolismo , Antibacterianos/farmacologiaRESUMO
Recombinant protein-based vaccines are a valid and safer alternative to traditional vaccines based on live-attenuated or killed pathogens. However, the immune response of subunit vaccines is generally lower compared to that elicited by traditional vaccines and usually requires the use of adjuvants. The use of self-assembling protein nanoparticles, as a platform for vaccine antigen presentation, is emerging as a promising approach to enhance the production of protective and functional antibodies. In this work we demonstrated the successful repetitive antigen display of the C-terminal ß-barrel domain of factor H binding protein, derived from serogroup B Meningococcus on the surface of different self-assembling nanoparticles using genetic fusion. Six nanoparticle scaffolds were tested, including virus-like particles with different sizes, geometries, and physicochemical properties. Combining computational and structure-based rational design we were able generate antigen-fused scaffolds that closely aligned with three-dimensional structure predictions. The chimeric nanoparticles were produced as recombinant proteins in Escherichia coli and evaluated for solubility, stability, self-assembly, and antigen accessibility using a variety of biophysical methods. Several scaffolds were identified as being suitable for genetic fusion with the ß-barrel from fHbp, including ferritin, a de novo designed aldolase from Thermotoga maritima, encapsulin, CP3 phage coat protein, and the Hepatitis B core antigen. In conclusion, a systematic screening of self-assembling nanoparticles has been applied for the repetitive surface display of a vaccine antigen. This work demonstrates the capacity of rational structure-based design to develop new chimeric nanoparticles and describes a strategy that can be utilized to discover new nanoparticle-based approaches in the search for vaccines against bacterial pathogens.
Assuntos
Vacinas Meningocócicas , Nanopartículas , Neisseria meningitidis , Aldeído Liases , Antígenos , Vacinas Bacterianas , Proteínas de Transporte , Fator H do Complemento , Ferritinas , Antígenos do Núcleo do Vírus da Hepatite B , Nanopartículas/química , Neisseria meningitidis/genética , Proteínas Recombinantes , Vacinas Combinadas , Vacinas de Subunidades AntigênicasRESUMO
Moraxella catarrhalis and nontypeable Haemophilus influenzae (NTHi) are pathogenic bacteria frequently associated with exacerbation of chronic obstructive pulmonary disease (COPD), whose hallmark is inflammatory oxidative stress. Neutrophils produce reactive oxygen species (ROS) which can boost antimicrobial response by promoting neutrophil extracellular traps (NET) and autophagy. Here, we showed that M. catarrhalis induces less ROS and NET production in differentiated HL-60 cells compared to NTHi. It is also able to actively interfere with these responses in chemically activated cells in a phagocytosis and opsonin-independent and contact-dependent manner, possibly by engaging host immunosuppressive receptors. M. catarrhalis subverts the autophagic pathway of the phagocytic cells and survives intracellularly. It also promotes the survival of NTHi which is otherwise susceptible to the host antimicrobial arsenal. In-depth understanding of the immune evasion strategies exploited by these two human pathogens could suggest medical interventions to tackle COPD and potentially other diseases in which they co-exist.
RESUMO
Both Gram-negative and Gram-positive pathogenic bacteria present a remarkable number of surface-exposed organelles and secreted toxins that allow them to control the primary stages of infection, bacterial attachment to host cell receptors and colonization. The mediators of these processes, called adhesins, form a heterogeneous group that varies in architecture, domain content and mechanism of binding. A full understanding of how adhesins mediate cellular adhesion and colonization requires quantitative functional assays to evaluate the strength of the binding interactions, as well as determination of the high-resolution three-dimensional structures of the molecules to provide the atomic details of the interactions. The combination of classical imaging techniques like X-ray crystallography and Nuclear Magnetic Resonance (NMR) with the emerging technique of single-particle electron cryomicroscopy has become a tremendously helpful tool to understand the three-dimensional structure at near atomic-level resolution of newly discovered adhesins and their complexes. A detailed study of the structure of these molecules, both isolated and expressed on bacterial surface is a fundamental requirement for understanding the adhesion mechanism to host cells. This chapter will focus on the structure determination of such surface-exposed protein structures in both Gram-negative and Gram-positive bacterial adhesins.
Assuntos
Adesinas Bacterianas/química , Adesinas Bacterianas/ultraestrutura , Microscopia Eletrônica/métodos , Fímbrias Bacterianas/química , Fímbrias Bacterianas/ultraestrutura , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/ultraestrutura , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/ultraestrutura , Humanos , Processamento de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador/tendências , Imageamento Tridimensional , Microscopia Eletrônica/tendências , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
GMMA, outer membrane vesicles resulting from hyperblebbing mutated bacterial strains, are a versatile vaccine platform for displaying both homologous and heterologous antigens. Periplasmic expression is a popular technique for protein expression in the lumen of the blebs. However, the ability of internalized antigens to induce antibody responses has not been extensively investigated. Herein, the Neisseria meningitidis factor H binding protein (fHbp) was heterologously expressed in the lumen of O-antigen positive (OAg+) and O-antigen negative (OAg-) Salmonella Typhimurium GMMA. Only the OAg- GMMA induced an anti-fHbp IgG response in mice if formulated on Alum, although it was weak and much lower compared to the recombinant fHbp. The OAg- GMMA on Alum showed partial instability, with possible exposure of fHbp to the immune system. When we chemically conjugated fHbp to the surface of both OAg+ and OAg- GMMA, these constructs induced a stronger functional response compared to the fHbp immunization alone. Moreover, the OAg+ GMMA construct elicited a strong response against both the target antigens (fHbp and OAg), with no immune interference observed. This result suggests that antigen localization on GMMA surface can play a critical role in the induction of an effective immune response and can encourage the development of GMMA based vaccines delivering key protective antigens on their surface.
RESUMO
Ensuring the stability of vaccines is crucial to successfully performing global immunization programs. Outer Membrane Vesicles (OMV) are receiving great attention as vaccine platforms. OMV are complex molecules and few data have been collected so far on their stability. OMV produced by bacteria, genetically modified to increase their spontaneous release, simplifying their production, are also known as Generalized Modules for Membrane Antigens (GMMA). We have performed accelerated stability studies on GMMA from different pathogens and verified the ability of physico-chemical and immunological methods to detect possible changes. High-temperature conditions (100 °C for 40 min) did not affect GMMA stability and immunogenicity in mice, in contrast to the effect of milder temperatures for a longer period of time (37 °C or 50 °C for 4 weeks). We identified critical quality attributes to monitor during stability assessment that could impact vaccine efficacy. In particular, specific recognition of antigens by monoclonal antibodies through competitive ELISA assays may replace in vivo tests for the potency assessment of GMMA-based vaccines.
RESUMO
A range of cationic delivery systems have been investigated as vaccine adjuvants, though few direct comparisons exist. To investigate the impact of the delivery platform, we prepared four cationic systems (emulsions, liposomes, polymeric nanoparticles and solid lipid nanoparticles) all containing equal concentrations of the cationic lipid dimethyldioctadecylammonium bromide in combination with the Neisseria adhesin A variant 3 subunit antigen. The formulations were physicochemically characterized and their ability to associate with cells and promote antigen processing (based on degradation of DQ-OVA, a substrate for proteases which upon hydrolysis is fluorescent) was compared in vitro and their vaccine efficacy (antigen-specific antibody responses and IFN-γ production) and biodistribution (antigen and adjuvant) were evaluated in vivo. Due to their cationic nature, all delivery systems gave high antigen loading (> 85%) with liposomes, lipid nanoparticles and emulsions being <200 nm, whilst polymeric nanoparticles were larger (~350 nm). In vitro, the particulate systems tended to promote cell uptake and antigen processing, whilst emulsions were less effective. Similarly, whilst the particulate delivery systems induced a depot (of both delivery system and antigen) at the injection site, the cationic emulsions did not. However, out of the systems tested the cationic emulsions induced the highest antibody responses. These results demonstrate that while cationic lipids can have strong adjuvant activity, their formulation platform influences their immunogenicity.